Biochip technology is a representative example of a new technology based on a combination of nanotechnology (NT), biotechnology (BT) and information technology (IT). Biochips are high-density microarrays comprising a variety of biomaterials on the surface of a solid support and can be divided, according to the kind of biomaterial attached to the solid support surface, into a DNA ship, a protein chip, a cell chip, a neuron chip and the like. Also, a combination of biochip technology with microfluidic technology enables the development of LOC (lab-on-a-chip) technology. Biochip technology includes a technique of immobilizing a biomaterial, a technique of making a solid support compatible with a biomaterial, a technique of making biomaterial microarrays, an assay technique of performing various biological reactions on a prepared chip, a technique of detecting reaction results, protein engineering of making a biomaterial to be immobilized, a gene recombination technique, and the like.
A protein chip, a kind of biochip, is a high-density microarray comprising a variety of proteins on a unit area of the surface of a solid support. In recent years, there have been efforts to fabricate protein chips using the principles and techniques for fabricating commercially available DNA chips. In general, commercially available DNA chips are mostly fabricated by immobilizing DNA on a glass substrate, the surface of which has been pretreated with a coating material. When a protein chip is fabricated using a method similar to a method used to fabricate a DNA chip, that is, when a protein chip is fabricated by immobilizing proteins on a glass substrate, the surface of which has been pretreated with a coating material, various problems arise due to the difference in physical and chemical properties between the target proteins to be immobilized.
Previous protein chips were produced by immobilizing proteins on a surface-treated glass substrate and used to perform a simple binding assay. The performance of the protein chip was determined by the activity of the immobilized protein and it was hard to work successfully (MacBeath and Schreiber, Science 289:1760, 2000). Such problems are caused by the denaturation, inactivation and degradation of proteins resulting from the difference in the inherent physical and chemical properties of proteins. In order to overcome these problems, studies have been conducted on surface treatment technology for immobilizing proteins suitable for protein characteristics which are distinguished from those of DNA and on materials for immobilizing protein. Such studies are focused on a method for performing immobilization on the surface of a protein chip while maintaining the activity of the protein. Examples thereof include Hydrogel™-coated slide (PerkinElmer), Versalinx chip (Prolinx), PDC chip which is a biochip commercially available from Zyomyx, etc.
Meanwhile, a sol-gel process is a technology which has been used to make a micro-structure by microprocessing. Particularly, it is a technology comprised of forming a binding net by a mild process and immobilizing biomolecules within the binding net by methods other than a covalent bonding method, instead of chemically attaching biomolecules to an inorganic material (Gill, I. and Ballesteros, A., Trends Biotechnol., 18:282, 2000).
Furthermore, many biomolecules, including enzymes, are immobilized on a mass sol-gel matrix and used to fabricate biocatalysts or biosensors (Reetz et al., Adv. Mater., 9:943, 1997). Particularly, these biomolecules are also used in the detection of optical color development due to their transparent optical properties (Edminston et al., J. Coll. Interf. Sci., 163:395, 1994). Also, biomolecules are known to be not only chemically but also thermally stabilized when they are immobilized on a sol-gel matrix (Dave et al., Anal. Chem., 66:1120, 1994).
In the case of biosensors, the sol-gel reaction is used as a method for forming and patterning a microstructure on a solid support as well as for simple immobilization. In this regard, the patterning method includes shaping a liquid-state sol using a mold by fluid dynamics, gelling the shaped material and removing the mold, thus forming a pattern. For example, a technology designated as micro-moduling in-capillaries (MIMIC) technology is a technique for patterning mesoscopic silica (Kim E, Xia Y, Whitesides G M. 1995. Polymer microstructures formed by moulding in capillaries. Nature 376:581-584; Marzolin et al., Adv. Mater. 10:571, 1998; Schuller et al., Appl. Optics 38:5799, 1999). This technology can be used in basic patterning of micro-fluid engineering.
However, since the activity of protein can be affected by various factors such as pH, it is important to set conditions for the maintenance of the activity by adding protein from its sol state in the sol-gel process. For this purpose, technologies of patterning a protein by premixing the protein with a sol using various mild conditions such as neutral pH (Kim et al., Biotechnol. Bioeng. 73:331 to 337, 2001) have been proposed, but there have been problems in that the sol-gel process rapidly progresses at neutral pH so that cracks may occur or the gel becomes opaque, according to the choice of additives. In addition, there has been a problem in that, because the pretreatment process of mixing the protein with a sol should be carried out, the concentration of spots is likely to be non-uniform.
In prior patents relating to sol-gel processes, there is a patent relating to a sol-gel biochip for improving the reactivity of a biomaterial, in which the sol-gel biochip is fabricated by a sol mixture containing the biomaterial is subjected to a gelation relation on a chip substrate so that the biomaterial is entrapped in the pores of the gel matrix and encapsulated by pores formed on the gel matrix. Also, there are patents relating to a method of fabricating a biochip using a sol-gel process, the method comprising screening a sol composition for sol-gel biochips, which prevents the modification of an immobilized biomaterial or increases the sensitivity of the biomaterial. However, there have been problems in that the fabrication method is complex and in that, in the process of preparing the sol composition, the activity of the biomaterial is reduced or the biomaterial is decomposed.
Accordingly, the present inventors have made many efforts to prevent the decrease in activity and the decomposition of a biomaterial during the preparation of a sol composition and, as a result, have found that, when a specific silicate monomer and additives are mixed sequentially in a specific order and dispensed onto a substrate or when these components are dispensed sequentially directly onto a substrate and gelled, the gelation rate thereof can be delayed compared to that in the conventional fabrication methods, thus making it possible to fabricate a significantly uniform biochip, and the decrease in activity and the decomposition of a biomaterial by the above components can be prevented, thus making it possible to fabricate a biochip having a very high sensitivity, thereby completing the present invention.